Our long-term OBJECTIVES remain to apply the fundamental concepts and broad technologies of cell and molecular biology to understand the role of cholangiocytes, the epithelial cells lining intrahepatic bile ducts, in health and disease. We continue to focus on the interactions between cholangiocytes and Cryptosporidium parvum (C. parvum), an emerging pathogen causing intestinal and biliary cryptosporidiosis. Recent evidence from our lab indicates that: (i) the cellular expression and regulation of key receptors [Toll-like receptors (TLRs)] and specific intracellular signaling pathways (NF-kB activation) are involved in cholangiocyte C. parvum recognition and strategic defense;and (ii) endogenous microRNA (miRNA)-mediated post- transcriptional gene regulation is involved in cholangiocyte defense responses to C. parvum infection. Thus, we will test the CENTRAL HYPOTHESIS that C. parvum-cholangiocyte interactions activate host-cell TLR/NF-kB signaling cascades initiating cholangiocyte defense responses including upregulation of both TLRs and anti-microbial peptides (defensins) through both transcriptional and post-transcriptional endogenous miRNA-mediated) regulatory pathways. In our three integrated SPECIFIC AIMS, we will test the hypotheses that: (i) cholangiocyte TLRs recognize C. parvum and modulate NF-kB activation resulting in upregulation of anti-microbial peptides (human beta-defensins [HBDs]) and TLRs through NF-kB mediated transcriptional regulation;(ii) miRNA-mediated post-transcriptional repression of TLR expression normally exists in cholangiocytes, is regulated by TLR/NF-kB signals, and is involved in C. parvum-induced upregulation of TLRs;and (iii) expression of TLRs and HBDs in cholangiocytes are required for eradication of C. parvum infection in the biliary tract in in vitro and in vivo experimental models. Innovative aspects of our program include: novel methodologies [small-interfering RNAs (siRNAs)], animal models (biliary cryptosporidiosis in wild type, TLR and MyD88 knock out mice), and new concepts (miRNA-mediated gene regulation in cholangiocyte defense). Thus, the experiments will define the molecular mechanisms by which C. parvum-cholangiocyte interactions trigger host epithelial defense, with an emphasis on the role of miRNA- mediated post-transcriptional regulation in TLR-associated defense, a NEW CONCEPT that may have important generalizability to both innate and adaptive immunity in health and disease.